Dietary flavonoids are known to have a variety of health benefits, ranging from anticancer, antioxidant, antiosteoperosis and cholesterol lowering to anti-aging. However, these compounds are poorly bioavailable which impedes the effort to develop them as drugs. The long-term goal of our study is to determine how coordinated interplay of key elements determines the overall disposition of flavonoids and contributes to their biological fate in vivo. The general hypothesis for the present research proposal is that the flavonoid bioavailability will be improved by interrupting interplays between key components of their disposition. Our previous research has shown that key components of flavonoid disposition are conjugating enzymes (i.e., sulfotransferases and UDP-glucuronosyltransferases) and efflux transporters (e.g., BCRP and MRP2). Our previous research also showed that flavonoids undergo the dual enteric and enterohepatic recycling processes, which increase their conjugate concentrations and apparent biological half- lives in vivo. The conjugated form of flavonoids may be bioavailable since local and systemic hydrolysis of conjugated flavonoids to aglycones by glucuronidases and sulfatases is a viable means of providing aglycone to target organs. Therefore, this present hypothesis is a step beyond the classical hypothesis that bioavailabilities are increased if aglycone AUC is increased. It is also a step beyond our original hypothesis that identification and inhibition of one key element in the disposition processes will lead to higher bioavailabilities, which we found to be untrue when we focused on identifying the enzyme isoform responsible. However, we did find that bioavailability of genistein improved in BCRP-/- (knockout) mice, although we observed a decrease in SULT activities as well. The Specific Aims of this continuing proposal are to: (1) determine the key UGT and SULT isoforms responsible for the metabolism of selected flavonoids, and key efflux transporters responsible for the excretion of phase II conjugates of flavonoids; (2) determine how key UGTs and relevant key efflux transporters identified in aim 1 compensate for each other's functional deficiency; (3) determine how key SULTs and relevant key efflux transporters identified in aim 1 compensate for each other's functional deficiency; and (4) determine how glucuronidation and sulfation pathways will compensate for each other's functional deficiency. Through these new studies, we seek to determine how interplay between key components of flavonoid metabolism can be effectively interrupted to improve their local and systematic bioavailabilities. The success of the proposed studies should lead to the development of dosage forms with improved bioavailability for humans. PUBLIC HEALTH RELEVANCE: Flavonoids have been shown to have a variety of beneficial effects based on in vitro studies using human cells and in vivo studies using animal models. However, there are no definitive clinical data to prove the effectiveness of this class of compounds in humans. A major impediment to definitive clinical studies of flavonoids lies with the facts that clinical trials are expensive (as the result of flavonoid's poor bioavailability) and there is no patentable dosage form that will attract private capital to enable the clinical trials. The proposed basic mechanistic studies should facilitate the development of new dosage forms with improved bioavailability, and benefit the health of American in the long run.